Protein synthesis via a microbial organism can be regulated mostly at the level of transcription and thus the selection of a most appropriate promoter that can strongly direct the synthesis of a desired protein is very important (Markrides, 1996, Microbial Reviews, 60, 512-538).
There are several factors that should be considered in selecting such a strong promoter. First, the promoter should have a very active transcriptional activity to synthesize sufficient amount of mRNA. Second, the promoter should be able to well control the protein expression, however, the promoter should not have any transcriptional activity or it should be kept at an extremely low level, if at all, prior to the induction of a given protein expression. Third, the promoter should be well transformed into a host cell. Finally, the promoter should have a relatively easy induction system for protein expression and is also preferred to be cost-effective.
There are a number of promoters that have been used in constructing recombinant expression vectors for protein biosynthesis using E. coli as a host cell; e.g., lac promoter (Roberts et al., 1979, Proc. Natl. Acad. Sci. USA, 76, 760-764), tac promoter (Aman et al., 1983, Gene, 25, 167-178), trc promoter (Brosius et al., 1985, J. Biol. Chem., 260, 3539-3541), PL or PR promoter (Elvin et al., 1990, Gene, 87, 123-126), and T7 promoter (Studier et al., 1986, J. Mol. Biol.;, 189, 113-130). These promoters are well known to exhibit very strong transcriptional activities in the presence of a particular inducer and accumulate more than 10-30% of the total proteins in cells. However, these promoters are considered disadvantageous in that their transcriptional activities are maintained at a relatively high level when cells are at a normal growth stage. Further, recombinant expression systems utilizing lac promoter or promoters derived from lambda phage are very effective and convenient in culturing E. coli for a general laboratory scale use, however, they are not well suited for production in the large culture. Still further, expression systems with lac promoter use isopropyl-β-D-thiogalactoside (IFTG) as an inducer, a highly expensive compound, and thus it becomes quite costly to prepare a large-scale cell culture. In case of an expression system using a promoter derived from lambda phage, it is required to increase a temperature for the expression of a protein and this increase in temperature results in generation of inactive inclusion bodies. Also, uniform temperature condition can be hardly maintained within a culture when preparing a large-scale culture.
Various efforts have been reported to solve the above-mentioned problems; e.g., phoA promoter (Miyake et al., 1985, J. Biochem., 97, 1429-436), cst-1 promoter (Turner et al., 1992, Biotechnol. Bioengin., 40, 271-79), nar promoter (Lee et al., 1996, Biotechnol. Lett., 18, 129-134), and trp promoter (Yansura et al., 1990, Methods. Enzymol., 185, 54-0). However, these promoters are not advantageous in that the regulation of expression is very complicated and inefficient.
Another important issue in recombinant protein expression might be a method of easy cultivation of host cells and easy induction of target proteins. One of such efforts is the use of modified specific medium for automatic induction (Studier, FW, 2005, Protein production by auto-induction in high density shaking cultures, Protein Expr Purif, 41(1):207-234). However, the promoters used were still strong ones of T7 and tac. Therefore, it has been an urgent need to develop a method for easy induction of automatic initiation of synthesis of target proteins.